Myeloid PHD2 Conditional Knockout Improves Intraplaque Angiogenesis and Vascular Remodeling in a Murine Model of Venous Bypass Grafting.

BACKGROUND: Intraplaque angiogenesis occurs in response to atherosclerotic plaque hypoxia, which is driven mainly by highly metabolically active macrophages. Improving plaque oxygenation by increasing macrophage hypoxic signaling, thus stimulating intraplaque angiogenesis, could restore cellular function and neovessel maturation, and decrease plaque formation. Prolyl hydroxylases (PHDs) regulate cellular responses to hypoxia. We therefore aimed to elucidate the role of myeloid PHD2, the dominant PHD isoform, on intraplaque angiogenesis in a murine model for venous bypass grafting. METHODS AND RESULTS: Myeloid PHD2 conditional knockout (PHD2cko) and PHD2 wild type mice on an Ldlr-/- background underwent vein graft surgery (n=11-15/group) by interpositioning donor caval veins into the carotid artery of genotype-matched mice. At postoperative day 28, vein grafts were harvested for morphometric and compositional analysis, and blood was collected for flow cytometry. Myeloid PHD2cko induced and improved intraplaque angiogenesis by improving neovessel maturation, which reduced intraplaque hemorrhage. Intima/media ratio was decreased in myeloid PHD2cko vein grafts. In addition, PHD2 deficiency prevented dissection of vein grafts and resulted in an increase in vessel wall collagen content. Moreover, the macrophage proinflammatory phenotype in the vein graft wall was attenuated in myeloid PHD2cko mice. In vitro cultured PHD2cko bone marrow-derived macrophages exhibited an increased proangiogenic phenotype compared with control. CONCLUSIONS: Myeloid PHD2cko reduces vein graft disease and ameliorates vein graft lesion stability by improving intraplaque angiogenesis.

Exploring the Effects of Human Bone Marrow-Derived Mononuclear Cells on Angiogenesis In Vitro.

Cell therapies involving the administration of bone marrow-derived mononuclear cells (BM-MNCs) for patients with chronic limb-threatening ischemia (CLTI) have shown promise; however, their overall effectiveness lacks evidence, and the exact mechanism of action remains unclear. In this study, we examined the angiogenic effects of well-controlled human bone marrow cell isolates on endothelial cells. The responses of endothelial cell proliferation, migration, tube formation, and aortic ring sprouting were analyzed in vitro, considering both the direct and paracrine effects of BM cell isolates. Furthermore, we conducted these investigations under both normoxic and hypoxic conditions to simulate the ischemic environment. Interestingly, no significant effect on the angiogenic response of human umbilical vein endothelial cells (HUVECs) following treatment with BM-MNCs was observed. This study fails to provide significant evidence for angiogenic effects from human bone marrow cell isolates on human endothelial cells. These in vitro experiments suggest that the potential benefits of BM-MNC therapy for CLTI patients may not involve endothelial cell angiogenesis.

Phosphorylcholine Monoclonal Antibody Therapy Decreases Intraplaque Angiogenesis and Intraplaque Hemorrhage in Murine Vein Grafts.

Phosphorylcholine (PC) is one of the main oxLDL epitopes playing a central role in atherosclerosis, due to its atherogenic and proinflammatory effects. PC can be cleared by natural IgM antibodies and low levels of these antibodies have been associated with human vein graft (VG) failure. Although PC antibodies are recognized for their anti-inflammatory properties, their effect on intraplaque angiogenesis (IPA) and intraplaque hemorrhage (IPH)-interdependent processes contributing to plaque rupture-are unknown. We hypothesized that new IgG phosphorylcholine antibodies (PC-mAb) could decrease vulnerable lesions in murine VGs.Therefore, hypercholesterolemic male ApoE3*Leiden mice received a (donor) caval vein interposition in the carotid artery and weekly IP injections of (5 mg/kg) PCmAb (n = 11) or vehicle (n = 12) until sacrifice at day 28. We found that PCmAb significantly decreased vein graft media (13%), intima lesion (25%), and increased lumen with 32% compared to controls. PCmAb increased collagen content (18%) and decreased macrophages presence (31%). PCmAb resulted in 23% decreased CD163+ macrophages content in vein grafts whereas CD163 expression was decreased in Hb:Hp macrophages. PCmAb significantly lowered neovessel density (34%), EC proliferation and migration with/out oxLDL stimulation. Moreover, PCmAb enhanced intraplaque angiogenic vessels maturation by increasing neovessel pericyte coverage in vivo (31%). Together, this resulted in a 62% decrease in IPH. PCmAb effectively inhibits murine atherosclerotic lesion formation in vein grafts by reducing IPA and IPH via decreased neovessel density and macrophages influx and increased neovessel maturation. PC-mAb therefore holds promise as a new therapeutic approach to prevent vein graft disease.

Extracellular vesicles enriched with an endothelial cell pro-survival microRNA affects skin tissue regeneration.

Endothelial cell (EC) activity is essential for tissue regeneration in several (patho)physiological contexts. However, our capacity to deliver in vivo biomolecules capable of controlling EC fate is relatively limited. Here, we screened a library of microRNA (miR) mimics and identified 25 miRs capable of enhancing the survival of ECs exposed to ischemia-mimicking conditions. In vitro, we showed that miR-425-5p, one of the hits, was able to enhance EC survival and migration. In vivo, using a mouse Matrigel plug assay, we showed that ECs transfected with miR-425-5p displayed enhanced survival compared with scramble-transfected ECs. Mechanistically, we showed that miR-425-5p modulated the PTEN/PI3K/AKT pathway and inhibition of miR-425-5p target genes (DACH1, PTEN, RGS5, and VASH1) phenocopied the pro-survival. For the in vivo delivery of miR-425-5p, we modulated small extracellular vesicles (sEVs) with miR-425-5p and showed, in vitro, that miR-425-5p-modulated sEVs were (1) capable of enhancing the survival of ECs exposed to ischemia-mimic conditions, and (2) efficiently internalized by skin cells. Finally, using a streptozotocin-induced diabetic wound healing mouse model, we showed that, compared with miR-scrambled-modulated sEVs, topical administration of miR-425-5p-modulated sEVs significantly enhanced wound healing, a process mediated by enhanced vascularization and skin re-epithelialization.

The Cortisol Response of Male and Female Choroidal Endothelial Cells: Implications for Central Serous Chorioretinopathy.

CONTEXT: Central serous chorioretinopathy (CSC) is a severe ocular disease characterized by fluid accumulation under the retina and abnormalities in the underlying vascular layer, the choroid. CSC has a striking prevalence in males of 80% to 90% of total patients. Corticosteroids are the most pronounced extrinsic risk factor for CSC. Choroidal endothelial cells (CECs) are important for the vascular integrity of the choroid, but the effects of corticosteroid effects in these cells are unknown. OBJECTIVE: We aimed to reveal the potential steroidal contribution to CSC. METHOD: We characterized the expression of the glucocorticoid, mineralocorticoid, and androgen receptor in the human choroid using immunohistochemistry. Using RNA-sequencing, we describe the cortisol response in human CECs derived from 5 male and 5 female postmortem donors. RESULTS: The glucocorticoid receptor was highly expressed in the human choroid, whereas no to minimal expression of the mineralocorticoid and androgen receptors was observed. The extensive transcriptional response to cortisol in human primary cultured CECs showed interindividual differences but very few sex differences. Several highly regulated genes such as ZBTB16 (log2 fold change males 7.9; females 6.2) provide strong links to choroidal vascular regulation. CONCLUSIONS: The glucocorticoid receptor predominantly mediates the response to cortisol in human CECs. Interindividual differences are an important determinant regarding the cortisol response in human cultured CECs, whereas intrinsic sex differences appear less pronounced. The marked response of particular target genes in endothelial cells to cortisol, such as ZBTB16, warrants further investigation into their potential role in the pathophysiology of CSC and other vascular conditions.

Interfering in the ALK1 Pathway Results in Macrophage-Driven Outward Remodeling of Murine Vein Grafts.

AIMS: Vein grafts are frequently used to bypass coronary artery occlusions. Unfortunately, vein graft disease (VGD) causes impaired patency rates. ALK1 mediates signaling by TGF-ß via TGFßR2 or BMP9/10 via BMPR2, which is an important pathway in fibrotic, inflammatory, and angiogenic processes in vascular diseases. The role of the TGF-ß pathway in VGD is previously reported, however, the contribution of ALK1 signaling is not known. Therefore, we investigated ALK1 signaling in VGD in a mouse model for vein graft disease using either genetic or pharmacological inhibition of the Alk1 signaling. METHODS AND RESULTS: Male ALK1 heterozygous (ALK1+/-), control C57BL/6, as well as hypercholesterolemic ApoE3*Leiden mice, underwent vein graft surgery. Histologic analyses of ALK1+/- vein grafts demonstrated increased outward remodeling and macrophage accumulation after 28 days. In hypercholesterolemic ApoE3*Leiden mice receiving weekly ALK1-Fc injections, ultrasound imaging showed 3-fold increased outward remodeling compared to controls treated with control-Fc, which was confirmed histologically. Moreover, ALK1-Fc treatment reduced collagen and smooth muscle cell accumulation, increased macrophages by 1.5-fold, and resulted in more plaque dissections. No difference was observed in intraplaque neovessel density. Flow cytometric analysis showed increased systemic levels of Ly6CHigh monocytes in ALK1-Fc treated mice, supported by in vitro increased MCP-1 and IL-6 production of LPS-stimulated and ALK1-Fc-treated murine monocytes and macrophages. CONCLUSION: Reduced ALK1 signaling in VGD promotes outward remodeling, increases macrophage influx, and promotes an unstable plaque phenotype. TRANSLATIONAL PERSPECTIVE: Vein graft disease (VGD) severely hampers patency rates of vein grafts, necessitating research of key disease-driving pathways like TGF-ß. The three-dimensional nature of VGD together with the multitude of disease driving factors ask for a comprehensive approach. Here, we combined in vivo ultrasound imaging, histological analyses, and conventional in vitro analyses, identifying the ambiguous role of reduced ALK1 signaling in vein graft disease. Reduced ALK1 signaling promotes outward remodeling, increases macrophage influx, and promotes an unstable plaque phenotype in murine vein grafts. Characterization of in vivo vascular remodeling over time is imperative to monitor VGD development and identify new therapies.

Periprocedural Hydrogen Sulfide Therapy Improves Vascular Remodeling and Attenuates Vein Graft Disease.

Background Failure rates after revascularization surgery remain high, both in vein grafts (VG) and arterial interventions. One promising approach to improve outcomes is endogenous upregulation of the gaseous transmitter-molecule hydrogen sulfide, via short-term dietary restriction. However, strict patient compliance stands as a potential translational barrier in the vascular surgery patient population. Here we present a new therapeutic approach, via a locally applicable gel containing the hydrogen sulfide releasing prodrug (GYY), to both mitigate graft failure and improve arterial remodeling. Methods and Results All experiments were performed on C57BL/6 (male, 12 weeks old) mice. VG surgery was performed by grafting a donor-mouse cava vein into the right common carotid artery of a recipient via an end-to-end anastomosis. In separate experiments arterial intimal hyperplasia was assayed via a right common carotid artery focal stenosis model. All mice were harvested at postoperative day 28 and artery/graft was processed for histology. Efficacy of hydrogen sulfide was first tested via GYY supplementation of drinking water either 1 week before VG surgery (pre-GYY) or starting immediately postoperatively (post-GYY). Pre-GYY mice had a 36.5% decrease in intimal/media+adventitia area ratio compared with controls. GYY in a 40% Pluronic gel (or vehicle) locally applied to the graft/artery had decreased intimal/media area ratios (right common carotid artery) and improved vessel diameters. GYY-geltreated VG had larger diameters at both postoperative days 14 and 28, and a 56.7% reduction in intimal/media+adventitia area ratios. Intimal vascular smooth muscle cell migration was decreased 30.6% after GYY gel treatment, which was reproduced in vitro. Conclusions Local gel-based treatment with the hydrogen sulfide-donor GYY stands as a translatable therapy to improve VG durability and arterial remodeling after injury.

bFGF blockade reduces intraplaque angiogenesis and macrophage infiltration in atherosclerotic vein graft lesions in ApoE3*Leiden mice.

Intraplaque angiogenesis increases the chance of unstable atherosclerotic plaque rupture and thrombus formation leading to myocardial infarction. Basic Fibroblast Growth Factor (bFGF) plays a key role in angiogenesis and inflammation and is involved in the pathogenesis of atherosclerosis. Therefore, we aim to test K5, a small molecule bFGF-inhibitor, on remodelling of accelerated atherosclerotic vein grafts lesions in ApoE3*Leiden mice. K5-mediated bFGF-signalling blockade strongly decreased intraplaque angiogenesis and intraplaque hemorrhage. Moreover, it reduced macrophage infiltration in the lesions by modulating CCL2 and VCAM1 expression. Therefore, K5 increases plaque stability. To study the isolated effect of K5 on angiogenesis and SMCs-mediated intimal hyperplasia formation, we used an in vivo Matrigel-plug mouse model that reveals the effects on in vivo angiogenesis and femoral artery cuff model to exclusively looks at SMCs. K5 drastically reduced in vivo angiogenesis in the matrigel plug model while no effect on SMCs migration nor proliferation could be seen in the femoral artery cuff model. Moreover, in vitro K5 impaired endothelial cells functions, decreasing migration, proliferation and tube formation. Our data show that K5-mediated bFGF signalling blockade in hypercholesterolemic ApoE3*Leiden mice reduces intraplaque angiogenesis, haemorrhage and inflammation. Therefore, K5 is a promising candidate to stabilize advanced atherosclerotic plaques.

Adenosine-to-Inosine Editing of Vasoactive MicroRNAs Alters Their Targetome and Function in Ischemia.

Adenosine-to-inosine (A-to-I) editing in the seed sequence of microRNAs can shift the microRNAs' targetomes and thus their function. Using public RNA-sequencing data, we identified 35 vasoactive microRNAs that are A-to-I edited. We quantified A-to-I editing of the primary (pri-)microRNAs in vascular fibroblasts and endothelial cells. Nine pri-microRNAs were indeed edited, and editing consistently increased under ischemia. We determined mature microRNA editing for the highest expressed microRNAs, i.e., miR-376a-3p, miR-376c-3p, miR-381-3p, and miR-411-5p. All four mature microRNAs were edited in their seed sequence. We show that both ADAR1 and ADAR2 (adenosine deaminase acting on RNA 1 and RNA 2) can edit pri-microRNAs in a microRNA-specific manner. MicroRNA editing also increased under ischemia in vivo in a murine hindlimb ischemia model and ex vivo in human veins. For each edited microRNA, we confirmed a shift in targetome. Expression of the edited microRNA targetomes, not the wild-type targetomes, was downregulated under ischemia in vivo. Furthermore, microRNA editing enhanced angiogenesis in vitro and ex vivo. In conclusion, we show that microRNA A-to-I editing is a widespread phenomenon, induced by ischemia. Each editing event results in a novel microRNA with a unique targetome, leading to increased angiogenesis.

Bis(maltolato)oxovanadium(IV) Induces Angiogenesis via Phosphorylation of VEGFR2.

VEGFR2 and VEGF-A play a pivotal role in the process of angiogenesis. VEGFR2 activation is regulated by protein tyrosine phosphatases (PTPs), enzymes that dephosphorylate the receptor and reduce angiogenesis. We aim to study the effect of PTPs blockade using bis(maltolato)oxovanadium(IV) (BMOV) on in vivo wound healing and in vitro angiogenesis. BMOV significantly improves in vivo wound closure by 45% in C57BL/6JRj mice. We found that upon VEGFR2 phosphorylation induced by endogenously produced VEGF-A, the addition of BMOV results in increased cell migration (45%), proliferation (40%) and tube formation (27%) in HUVECs compared to control. In a mouse ex vivo, aortic ring assay BMOV increased the number of sprouts by 3 folds when compared to control. However, BMOV coadministered with exogenous VEGF-A increased ECs migration, proliferation and tube formation by only 41%, 18% and 12% respectively and aortic ring sprouting by only 1-fold. We also found that BMOV enhances VEGFR2 Y951 and p38MAPK phosphorylation, but not ERK1/2. The level of phosphorylation of these residues was the same in the groups treated with BMOV supplemented with exogenous VEGF-A and exogenous VEGF-A only. Our study demonstrates that BMOV is able to enhance wound closure in vivo. Moreover, in the presence of endogenous VEGF-A, BMOV is able to stimulate in vitro angiogenesis by increasing the phosphorylation of VEGFR2 and its downstream proangiogenic enzymes. Importantly, BMOV had a stronger proangiogenic effect compared to its effect in coadministration with exogenous VEGF-A.

Prolonged Hyperoxygenation Treatment Improves Vein Graft Patency and Decreases Macrophage Content in Atherosclerotic Lesions in ApoE3*Leiden Mice.

Unstable atherosclerotic plaques frequently show plaque angiogenesis which increases the chance of rupture and thrombus formation leading to infarctions. Hypoxia plays a role in angiogenesis and inflammation, two processes involved in the pathogenesis of atherosclerosis. We aim to study the effect of resolution of hypoxia using carbogen gas (95% O2, 5% CO2) on the remodeling of vein graft accelerated atherosclerotic lesions in ApoE3*Leiden mice which harbor plaque angiogenesis. Single treatment resulted in a drastic decrease of intraplaque hypoxia, without affecting plaque composition. Daily treatment for three weeks resulted in 34.5% increase in vein graft patency and increased lumen size. However, after three weeks intraplaque hypoxia was comparable to the controls, as were the number of neovessels and the degree of intraplaque hemorrhage. To our surprise we found that three weeks of treatment triggered ROS accumulation and subsequent Hif1a induction, paralleled with a reduction in the macrophage content, pointing to an increase in lesion stability. Similar to what we observed in vivo, in vitro induction of ROS in bone marrow derived macrophages lead to increased Hif1a expression and extensive DNA damage and apoptosis. Our study demonstrates that carbogen treatment did improve vein graft patency and plaque stability and reduced intraplaque macrophage accumulation via ROS mediated DNA damage and apoptosis but failed to have long term effects on hypoxia and intraplaque angiogenesis.

CD8+ T Cells Protect During Vein Graft Disease Development.

Aims: Vein grafts are frequently used conduits for arterial reconstruction in patients with cardiovascular disease. Unfortunately, vein graft disease (VGD) causes diminished patency rates. Innate immune system components are known to contribute to VGD. However, the role of T cells has yet to be established. The purpose of this study was to investigate the role of T cells and T cell activation pathways via the T cell receptor (TCR), co-stimulation and bystander effect in VGD. Methods and results: Here, we show upon vein graft surgery in mice depleted of CD4+ T cells or CD8+ T cells, that CD8+ T cells are locally activated and have a major protective role for vein graft patency. In presence of CD8+ T cells vein grafts appear patent while CD8+ T cell depletion results in occluded vein grafts with increases apoptosis. Importantly, the protective effect of CD8+ T cells in VGD development was TCR and co-stimulation independent. This was demonstrated in vein grafts of OT-I mice, CD70-/-, CD80/86-/-, and CD70/80/86-/- mice compared to C57BL/6 mice. Interestingly, cytokines including IL-15, IL-18, IL-33, and TNF are up-regulated in vein grafts. These cytokines are co-operatively capable to activate CD8+ T cells in a bystander-mediated fashion, in contrast to CD4+ T cells. Conclusions: T cells are modulators of VGD with a specific protective role of CD8+ T cells, which are locally activated in vein grafts. CD8+ T cells may protect against occlusive lesions by providing survival signals, and concert their protection independent of TCR and co-stimulation signaling.

IRF3 and IRF7 mediate neovascularization via inflammatory cytokines.

OBJECTIVE: To elucidate the role of interferon regulatory factor (IRF)3 and IRF7 in neovascularization. METHODS: Unilateral hind limb ischaemia was induced in Irf3-/- , Irf7-/- and C57BL/6 mice by ligation of the left common femoral artery. Post-ischaemic blood flow recovery in the paw was measured with laser Doppler perfusion imaging. Soleus, adductor and gastrocnemius muscles were harvested to investigate angiogenesis and arteriogenesis and inflammation. RESULTS: Post-ischaemic blood flow recovery was decreased in Irf3-/- and Irf7-/- mice compared to C57BL/6 mice at all time points up to and including sacrifice, 28 days after surgery (t28). This was supported by a decrease in angiogenesis and arteriogenesis in soleus and adductor muscles of Irf3-/- and Irf7-/- mice at t28. Furthermore, the number of macrophages around arterioles in adductor muscles was decreased in Irf3-/- and Irf7-/- mice at t28. In addition, mRNA expression levels of pro-inflammatory cytokines (tnfα, il6, ccl2) and growth factor receptor (vegfr2), were decreased in gastrocnemius muscles of Irf3-/- and Irf7-/- mice compared to C57BL/6 mice. CONCLUSION: Deficiency of IRF3 and IRF7 results in impaired post-ischaemic blood flow recovery caused by attenuated angiogenesis and arteriogenesis linked to a lack of inflammatory components in ischaemic tissue. Therefore, IRF3 and IRF7 are essential regulators of neovascularization.

The Effect of Corticosteroids on Human Choroidal Endothelial Cells: A Model to Study Central Serous Chorioretinopathy.

Purpose: To isolate, culture, and characterize primary human choroidal endothelial cells, and to assess their responsiveness to corticosteroids, in order to enable knowledge gain on the pathogenesis of central serous chorioretinopathy. Methods: Choroidal endothelial cells were isolated from cadaveric human donors. Magnetic-activated cell sorting with anti-human CD31 was performed for choroidal endothelial cell isolation. Primary cultures of purified choroidal endothelial cells were treated with several regimens of corticosteroids and analyzed for effects on primary corticosteroid responsive genes. Results: Isolated choroidal endothelial cell cultures had a cobblestone appearance in monolayer cultures and stained positive for vascular endothelial cadherin. Moreover, on a 3D-Matrigel matrix, these cells formed capillary-like structures, characteristic of in vitro endothelial cells. Primary cultures of purified choroidal endothelial cells treated with several regimens of corticosteroids demonstrated significant transcriptional upregulation of primary corticosteroid responsive genes (FKBP5, PER1, GILZ, and SGK1). Further pharmacologic analysis using specific agonists (dexamethasone, aldosterone) and antagonists (mifepristone, spironolactone) for either the glucocorticoid receptor or the mineralocorticoid receptor showed that this response was exclusively mediated by the glucocorticoid receptor in our model. Conclusions: With this optimized choroidal endothelial cell isolation and culturing protocol, we have established an in vitro model that appears very suitable for research on both central serous chorioretinopathy and other diseases in which corticosteroids and choroidal endothelial cells are involved. Our model proves to be suitable for studying effects mediated through the glucocorticoid receptor. The role of mineralocorticoid receptor-mediated effects needs further research, both in vivo and in cell model development.

Adenosine-to-Inosine Editing of MicroRNA-487b Alters Target Gene Selection After Ischemia and Promotes Neovascularization.

RATIONALE: Adenosine-to-inosine editing of microRNAs has the potential to cause a shift in target site selection. 2'-O-ribose-methylation of adenosine residues, however, has been shown to inhibit adenosine-to-inosine editing. OBJECTIVE: To investigate whether angiomiR miR487b is subject to adenosine-to-inosine editing or 2'-O-ribose-methylation during neovascularization. METHODS AND RESULTS: Complementary DNA was prepared from C57BL/6-mice subjected to hindlimb ischemia. Using Sanger sequencing and endonuclease digestion, we identified and validated adenosine-to-inosine editing of the miR487b seed sequence. In the gastrocnemius muscle, pri-miR487b editing increased from 6.7±0.4% before to 11.7±1.6% (P=0.02) 1 day after ischemia. Edited pri-miR487b is processed into a novel microRNA, edited miR487b, which is also upregulated after ischemia. We confirmed editing of miR487b in multiple human primary vascular cell types. Short interfering RNA-mediated knockdown demonstrated that editing is adenosine deaminase acting on RNA 1 and 2 dependent. Using reverse-transcription at low dNTP concentrations followed by quantitative-PCR, we found that the same adenosine residue is methylated in mice and human primary cells. In the murine gastrocnemius, the estimated methylation fraction increased from 32.8±14% before to 53.6±12% 1 day after ischemia. Short interfering RNA knockdown confirmed that methylation is fibrillarin dependent. Although we could not confirm that methylation directly inhibits editing, we do show that adenosine deaminase acting on RNA 1 and 2 and fibrillarin negatively influence each other's expression. Using multiple luciferase reporter gene assays, we could demonstrate that editing results in a complete switch of target site selection. In human primary cells, we confirmed the shift in miR487b targeting after editing, resulting in a edited miR487b targetome that is enriched for multiple proangiogenic pathways. Furthermore, overexpression of edited miR487b, but not wild-type miR487b, stimulates angiogenesis in both in vitro and ex vivo assays. CONCLUSIONS: MiR487b is edited in the seed sequence in mice and humans, resulting in a novel, proangiogenic microRNA with a unique targetome. The rate of miR487b editing, as well as 2'-O-ribose-methylation, is increased in murine muscle tissue during postischemic neovascularization. Our findings suggest miR487b editing plays an intricate role in postischemic neovascularization.

The epigenetic factor PCAF regulates vascular inflammation and is essential for intimal hyperplasia development.

OBJECTIVE: Genetic P300/CBP-associated factor (PCAF) variation affects restenosis-risk in patients. PCAF has lysine acetyltransferase activity and promotes nuclear factor kappa-beta (NFκB)-mediated inflammation, which drives post-interventional intimal hyperplasia development. We studied the contributing role of PCAF in post-interventional intimal hyperplasia. METHODS AND RESULTS: PCAF contribution to inflammation and intimal hyperplasia was assessed in leukocytes, macrophages and vascular smooth muscle cells (vSMCs) in vitro and in a mouse model for intimal hyperplasia, in which a cuff is placed around the femoral artery. PCAF deficiency downregulate CCL2, IL-6 and TNF-alpha expression, as demonstrated on cultured vSMCs, leukocytes and macrophages. PCAF KO mice showed a 71.8% reduction of vSMC-rich intimal hyperplasia, a 73.4% reduction of intima/media ratio and a 63.7% reduction of luminal stenosis after femoral artery cuff placement compared to wild type (WT) mice. The association of PCAF and vascular inflammation was further investigated using the potent natural PCAF inhibitor garcinol. Garcinol treatment reduced CCL2 and TNF-alpha expression, as demonstrated on cultured vSMCs and leukocytes. To assess the effect of garcinol treatment on vascular inflammation we used hypercholesterolemic ApoE*3-Leiden mice. After cuff placement, garcinol treatment resulted in reduced arterial leukocyte and macrophage adherence and infiltration after three days compared to untreated animals. CONCLUSIONS: These results identify a vital role for the lysine acetyltransferase PCAF in the regulation of local inflammation after arterial injury and likely the subsequent vSMC proliferation, responsible for intimal hyperplasia.

Short hairpin RNA gene silencing of prolyl hydroxylase-2 with a minicircle vector improves neovascularization of hindlimb ischemia.

In this study, we target the hypoxia inducible factor-1 alpha (HIF-1-alpha) pathway by short hairpin RNA interference therapy targeting prolyl hydroxylase-2 (shPHD2). We use the minicircle (MC) vector technology as an alternative for conventional nonviral plasmid (PL) vectors in order to improve neovascularization after unilateral hindlimb ischemia in a murine model. Gene expression and transfection efficiency of MC and PL, both in vitro and in vivo, were assessed using bioluminescence imaging (BLI) and firefly luciferase (Luc) reporter gene. C57Bl6 mice underwent unilateral electrocoagulation of the femoral artery and gastrocnemic muscle injection with MC-shPHD2, PL-shPHD2, or phosphate-buffered saline (PBS) as control. Blood flow recovery was monitored using laser Doppler perfusion imaging, and collaterals were visualized by immunohistochemistry and angiography. MC-Luc showed a 4.6-fold higher in vitro BLI signal compared with PL-Luc. BLI signals in vivo were 4.3×10(5)±3.3×10(5) (MC-Luc) versus 0.4×10(5)±0.3×10(5) (PL-Luc) at day 28 (p=0.016). Compared with PL-shPHD2 or PBS, MC-shPHD2 significantly improved blood flow recovery, up to 50% from day 3 until day 14 after ischemia induction. MC-shPHD2 significantly increased collateral density and capillary density, as monitored by alpha-smooth muscle actin expression and CD31(+) expression, respectively. Angiography data confirmed the histological findings. Significant downregulation of PHD2 mRNA levels by MC-shPHD2 was confirmed by quantitative polymerase chain reaction. Finally, Western blot analysis confirmed significantly higher levels of HIF-1-alpha protein by MC-shPHD2, compared with PL-shPHD2 and PBS. This study provides initial evidence of a new potential therapeutic approach for peripheral artery disease. The combination of HIF-1-alpha pathway targeting by shPHD2 with the robust nonviral MC plasmid improved postischemic neovascularization, making this approach a promising potential treatment option for critical limb ischemia.